Fabaeformiscandona kushiroensis Hiruta & Hiruta

Hiruta, Shimpei F. & Hiruta, Shin-ichi, 2015, Description of a species of Fabaeformiscandona (Ostracoda, Crustacea) from Kushiro Marsh, Hokkaido, Japan, with the nearly complete mitochondrial genomic sequence, Biodiversity Data Journal 3, pp. 7074-7074 : 7074

Fabaeformiscandona kushiroensis Hiruta & Hiruta sp. n.

Materials

Type status: Holotype. Occurrence: catalogNumber: ICHUM_5034 ; recordedBy: Shimpei F. Hiruta; individualCount: 1; sex: male; lifeStage: adult; preparations: soft parts mounted on 10 slides, carapace mounted on a microfossil slide; Taxon: scientificName: Fabaeformiscandonakushiroensis; Location: country: Japan; stateProvince: Hokkaido; locality: Kushiro Marsh National Park, Hokkaido, Japan ; verbatimElevation: 10 m; verbatimCoordinates: 43°6′17.6″N, 144°19′46.5″E; decimalLatitude: 43.105; decimalLongitude: 144.3291; georeferenceProtocol: GPS; Identification: identifiedBy: Shimpei F. Hiruta; dateIdentified: 2013; Event: samplingProtocol: Strained by 0.1 mm mesh sieve; eventDate: 12/27/2012; Record Level: language: en; collectionID: ICHUM 5034; institutionCode: Invertebrate Collection of the Hokkaido University Museum (ICHUM); collectionCode: Ostracoda; basisOfRecord: PreservedSpecimen

Type status: Paratype. Occurrence: catalogNumber: ICHUM_5035 ; recordedBy: Shimpei F. Hiruta; individualCount: 1; sex: female; lifeStage: adult; preparations: soft parts mounted on 8 slides, carapaces mounted on a microfossil slide.; Taxon: scientificName: Fabaeformiscandonakushiroensis; Location: country: Japan; stateProvince: Hokkaido; locality: Kushiro Marsh National Park, Hokkaido, Japan ; verbatimElevation: 10 m; verbatimCoordinates: 43°6′17.6″N, 144°19′46.5″E; decimalLatitude: 43.105; decimalLongitude: 144.3291; georeferenceProtocol: GPS; Identification: identifiedBy: Shimpei F. Hiruta; dateIdentified: 2013; Event: samplingProtocol: Strained by 0.1 mm mesh sieve; eventDate: 12/27/2012; Record Level: language: en; collectionID: ICHUM 5035; institutionCode: Invertebrate Collection of the Hokkaido University Museum (ICHUM); collectionCode: Ostracoda; basisOfRecord: PreservedSpecimen

Type status: Paratype. Occurrence: catalogNumber: ICHUM_5036 ; recordedBy: Shimpei F. Hiruta; individualCount: 1; sex: male; lifeStage: adult; preparations: mounted on a slide; Taxon: scientificName: Fabaeformiscandonakushiroensis; Location: country: Japan; stateProvince: Hokkaido; locality: Kushiro Marsh National Park, Hokkaido, Japan ; verbatimElevation: 10 m; verbatimCoordinates: 43°6′17.6″N, 144°19′46.5″E; decimalLatitude: 43.105; decimalLongitude: 144.3291; georeferenceProtocol: GPS; Identification: identifiedBy: Shimpei F. Hiruta; dateIdentified: 2013; Event: samplingProtocol: Strained by 0.1 mm mesh sieve; eventDate: 12/27/2012; Record Level: language: en; collectionID: ICHUM 5036; institutionCode: Invertebrate Collection of the Hokkaido University Museum (ICHUM); collectionCode: Ostracoda; basisOfRecord: PreservedSpecimen

Type status: Paratype. Occurrence: catalogNumber: ICHUM_5037 ; recordedBy: Shimpei F. Hiruta; individualCount: 1; sex: male; lifeStage: adult; preparations: mounted on a slide; Taxon: scientificName: Fabaeformiscandonakushiroensis; Location: country: Japan; stateProvince: Hokkaido; locality: Kushiro Marsh National Park, Hokkaido, Japan ; verbatimElevation: 10 m; verbatimCoordinates: 43°6′17.6″N, 144°19′46.5″E; decimalLatitude: 43.105; decimalLongitude: 144.3291; georeferenceProtocol: GPS; Identification: identifiedBy: Shimpei F. Hiruta; dateIdentified: 2013; Event: samplingProtocol: Strained by 0.1 mm mesh sieve; eventDate: 12/27/2012; Record Level: language: en; collectionID: ICHUM 5037; institutionCode: Invertebrate Collection of the Hokkaido University Museum (ICHUM); collectionCode: Ostracoda; basisOfRecord: PreservedSpecimen

Type status: Paratype. Occurrence: catalogNumber: ICHUM_5038 ; recordedBy: Shimpei F. Hiruta; individualCount: 1; sex: female; lifeStage: adult; preparations: mounted on a slide; Taxon: scientificName: Fabaeformiscandonakushiroensis; Location: country: Japan; stateProvince: Hokkaido; locality: Kushiro Marsh National Park, Hokkaido, Japan ; verbatimElevation: 10 m; verbatimCoordinates: 43°6′17.6″N, 144°19′46.5″E; decimalLatitude: 43.105; decimalLongitude: 144.3291; georeferenceProtocol: GPS; Identification: identifiedBy: Shimpei F. Hiruta; dateIdentified: 2013; Event: samplingProtocol: Strained by 0.1 mm mesh sieve; eventDate: 12/27/2012; Record Level: language: en; collectionID: ICHUM 5038; institutionCode: Invertebrate Collection of the Hokkaido University Museum (ICHUM); collectionCode: Ostracoda; basisOfRecord: PreservedSpecimen

Type status: Paratype. Occurrence: catalogNumber: ICHUM_5039 ; recordedBy: Shimpei F. Hiruta; individualCount: 1; sex: male; lifeStage: adult; preparations: mounted on a stub for SEM observation; Taxon: scientificName: Fabaeformiscandonakushiroensis; Location: country: Japan; stateProvince: Hokkaido; locality: Kushiro Marsh National Park, Hokkaido, Japan ; verbatimElevation: 10 m; verbatimCoordinates: 43°6′17.6″N, 144°19′46.5″E; decimalLatitude: 43.105; decimalLongitude: 144.3291; georeferenceProtocol: GPS; Identification: identifiedBy: Shimpei F. Hiruta; dateIdentified: 2013; Event: samplingProtocol: Strained by 0.1 mm mesh sieve; eventDate: 12/27/2012; Record Level: language: en; collectionID: ICHUM 5039; institutionCode: Invertebrate Collection of the Hokkaido University Museum (ICHUM); collectionCode: Ostracoda; basisOfRecord: PreservedSpecimen

Type status: Paratype. Occurrence: catalogNumber: ICHUM_5040 ; recordedBy: Shimpei F. Hiruta; individualCount: 1; sex: female; lifeStage: adult; preparations: mounted on a stub for SEM observation; Taxon: scientificName: Fabaeformiscandonakushiroensis; Location: country: Japan; stateProvince: Hokkaido; locality: Kushiro Marsh National Park, Hokkaido, Japan ; verbatimElevation: 10 m; verbatimCoordinates: 43°6′17.6″N, 144°19′46.5″E; decimalLatitude: 43.105; decimalLongitude: 144.3291; georeferenceProtocol: GPS; Identification: identifiedBy: Shimpei F. Hiruta; dateIdentified: 2013; Event: samplingProtocol: Strained by 0.1 mm mesh sieve; eventDate: 12/27/2012; Record Level: language: en; collectionID: ICHUM 5040; institutionCode: Invertebrate Collection of the Hokkaido University Museum (ICHUM); collectionCode: Ostracoda; basisOfRecord: PreservedSpecimen

Type status: Paratype. Occurrence: catalogNumber: ICHUM_5041 ; recordedBy: Shimpei F. Hiruta; individualCount: 1; sex: female; lifeStage: adult; preparations: mounted on a stub for SEM observation; Taxon: scientificName: Fabaeformiscandonakushiroensis; Location: country: Japan; stateProvince: Hokkaido; locality: Kushiro Marsh National Park, Hokkaido, Japan ; verbatimElevation: 10 m; verbatimCoordinates: 43°6′17.6″N, 144°19′46.5″E; decimalLatitude: 43.105; decimalLongitude: 144.3291; georeferenceProtocol: GPS; Identification: identifiedBy: Shimpei F. Hiruta; dateIdentified: 2013; Event: samplingProtocol: Strained by 0.1 mm mesh sieve; eventDate: 12/27/2012; Record Level: language: en; collectionID: ICHUM 5041; institutionCode: Invertebrate Collection of the Hokkaido University Museum (ICHUM); collectionCode: Ostracoda; basisOfRecord: PreservedSpecimen

Type status: Paratype. Occurrence: catalogNumber: ICHUM_5042 ; recordedBy: Shimpei F. Hiruta; individualCount: 1; sex: female; lifeStage: adult; preparations: mounted on a stub for SEM observation; Taxon: scientificName: Fabaeformiscandonakushiroensis; Location: country: Japan; stateProvince: Hokkaido; locality: Kushiro Marsh National Park, Hokkaido, Japan ; verbatimElevation: 10 m; verbatimCoordinates: 43°6′17.6″N, 144°19′46.5″E; decimalLatitude: 43.105; decimalLongitude: 144.3291; georeferenceProtocol: GPS; Identification: identifiedBy: Shimpei F. Hiruta; dateIdentified: 2013; Event: samplingProtocol: Strained by 0.1 mm mesh sieve; eventDate: 12/27/2012; Record Level: language: en; collectionID: ICHUM 5042; institutionCode: Invertebrate Collection of the Hokkaido University Museum (ICHUM); collectionCode: Ostracoda; basisOfRecord: PreservedSpecimen

Type status: Paratype. Occurrence: catalogNumber: ICHUM_5043 ; recordedBy: Shimpei F. Hiruta; individualCount: 1; sex: male; lifeStage: adult; preparations: exoskeleton mounted on a slide after DNA extraction; associatedSequences: GenBank: AP014656, AB996740; Taxon: scientificName: Fabaeformiscandonakushiroensis; Location: country: Japan; stateProvince: Hokkaido; locality: Kushiro Marsh National Park, Hokkaido, Japan ; verbatimElevation: 10 m; verbatimCoordinates: 43°6′17.6″N, 144°19′46.5″E; decimalLatitude: 43.105; decimalLongitude: 144.3291; georeferenceProtocol: GPS; Identification: identifiedBy: Shimpei F. Hiruta; dateIdentified: 2013; Event: samplingProtocol: Strained by 0.1 mm mesh sieve; eventDate: 12/27/2012; Record Level: language: en; collectionID: ICHUM 5043; institutionCode: Invertebrate Collection of the Hokkaido University Museum (ICHUM); collectionCode: Ostracoda; basisOfRecord: PreservedSpecimen

Type status: Paratype. Occurrence: catalogNumber: ICHUM_5044 ; recordedBy: Shimpei F. Hiruta; individualCount: 50; sex: undetermined; lifeStage: adult; preparations: exoskeleton preserved in 99% ethanol after DNA extraction; Taxon: scientificName: Fabaeformiscandonakushiroensis; Location: country: Japan; stateProvince: Hokkaido; locality: Kushiro Marsh National Park, Hokkaido, Japan ; verbatimElevation: 10 m; verbatimCoordinates: 43°6′17.6″N, 144°19′46.5″E; decimalLatitude: 43.105; decimalLongitude: 144.3291; georeferenceProtocol: GPS; Identification: identifiedBy: Shimpei F. Hiruta; dateIdentified: 2013; Event: samplingProtocol: Strained by 0.1 mm mesh sieve; eventDate: 12/27/2012; Record Level: language: en; collectionID: ICHUM 5044; institutionCode: Invertebrate Collection of the Hokkaido University Museum (ICHUM); collectionCode: Ostracoda; basisOfRecord: PreservedSpecimen

Type status: Paratype. Occurrence: catalogNumber: ICHUM_5116 ; recordedBy: Shimpei F. Hiruta; individualCount: 3; sex: 1 male, 2 females; lifeStage: adult; preparations: mounted on a stub for SEM observation; Taxon: scientificName: Fabaeformiscandonakushiroensis; Location: country: Japan; stateProvince: Hokkaido; locality: Kushiro Marsh National Park, Hokkaido, Japan ; verbatimElevation: 10 m; verbatimCoordinates: 43°6′17.6″N, 144°19′46.5″E; decimalLatitude: 43.105; decimalLongitude: 144.3291; georeferenceProtocol: GPS; Identification: identifiedBy: Shimpei F. Hiruta; dateIdentified: 2013; Event: samplingProtocol: Strained by 0.1 mm mesh sieve; eventDate: 12/27/2012; Record Level: language: en; collectionID: ICHUM 5116; institutionCode: Invertebrate Collection of the Hokkaido University Museum (ICHUM); collectionCode: Ostracoda; basisOfRecord: PreservedSpecimen

Description

Description of male.

Carapace (Figs 2, 3, 7) 1.16-1.24 mm long, 0.62-0.66 mm high (n = 4; holotype 1.24 mm long, 0.66 mm high); greatest height situated at dorsal hump approximately one-third of length from posterior margin; either side of the dorsal humps almost straight; anterior and posterior margins equally rounded; ventral margin concave at two-fifths of length from anterior end. Calcified inner lamella wider anteriorly than posteriorly. Carapace laterally compressed in dorsal view; greatest width situated about at mid-length; LV overlapping to RV; postero-dorsal flap of LV broad and somewhat longer than antero-dorsal flap (Fig. 3; Suppl. material 4). Surface of valves smooth, color translucent white (Fig. 2).

A1 (Fig. 9) seven articulated segmented. First two podomeres fused, with two dorsal setae and two long, stout apico-ventral setae. Third podomere rectangular, with apico-dorsal seta. Fourth podomere quadrate, with one apico-dorsal seta. Fifth podomere quadrate, with one long apico-dorsal and one apico-ventral setae. Sixth podomere rectangular, with two long apico-dorsal and one apico-ventral setae. Seventh podomere elongate, with one long apico-dorsal and two apico-ventral setae. Eighth podomere elongate, slender, with two long and one shorter setae, and aesthetasc ya.

A2 (Fig. 10) five-segmented. First podomere (Pr) with one apico-ventral seta of medium length; one long antero-proximal plumose seta; one long antero-proximal seta; and one long latero-proximal seta. Second podomere (EI) with one apico-ventral seta, aesthetasc Y, and exopodite (Exo); exopodite consists of one long distally plumed and two short setae. Third podomere (EII) with two mid-apical male bristles (t2 and t3), one short apico-ventral (t1) and one apico-dorsal (t4) setae, and aesthetasc y2. Fourth podomere (EIII) with G1, short G2, z1, and z2 claws, and G3 and z3 setae. Fifth podomere (EIV) with GM and shorter GM claws, one apical seta, and aesthetasc y3.

Md (Fig. 11) consists of coxal plate and four-segmented palp. Coxal plate with antero-lateral seta and seven stout teeth, latter interspersed with several setae of various lengths. First podomere of palp with exopodal plate (Exo) and one long and one short, stout inner-distal plumose setae, one long antero-distal seta, and alpha simple seta. Second podomere of palp with beta seta, group of four setae, and one plumose seta. Third podomere of palp with three outer-apical setae, one mid-apical gamma seta, and two long apical setae. Fourth podomere of palp with two claws and two apical setae.

Mx (Figs 12, 13) with elongate vibratory plate, three masticatory processes, and two-segmented palp. Masticatory process with numerous setae. First podomere with three apical plumose setae.

L5 (Figs 14, 15) with palp, two filament-like setae (Exo), one antero-proximal seta, one antero-apical seta, and one postero-apical seta. Palp transformed into asymmetrical clasping processes; left clasping process (Fig. 14) slender, somewhat curved and hook-like in shape, with two short mid-setae; right clasping process (Fig. 15) sharply curved and proximally stout, helmet-like in shape, with two short mid-setae. Masticatory process with numerous setae.

L6 (Fig. 16) five-segmented. Apical setae (e, f, g) on second to fourth podomeres long, each extending past end of subsequent podomere. Terminal claw (h2) slender, long.

L7 (Fig. 17) five-segmented. Penultimate segment subdivided. First podomere with two setae (d1, dp). Fourth podomere with one apical seta (g). Fifth podomere with two long (h2, h3) and one shorter (h1) setae. One third of distal tips of the h3 seta bend.

Uropodal ramus (Fig. 18) with short anterior seta (Sa) and well-developed posterior seta (Sp) longer than terminal claws. Two terminal claws simple, slender.

Hp (Fig. 19) distally with three lobes distally; median lobe (h) elongate, directed toward dorsal side, truncate distally; outer lobe (a) elongate, directed dorsally acute distally. Inner lobe (b) largely rounded and overlapping to the other two lobes. M-process well developed, S-shaped, rounded distally. Bursa copulatrix well-developed, pointed distally, with proximal bump.

Zenker’s organ (Fig. 20) with 5 + 2 internal rings of spines. Seminal vesicle located at anterior end.

Description of female.

Carapace (Figs 4, 5, 6, 8) 1.07-1.14 mm long, 0.58-0.59 mm high (n = 5; allotype 1.07 mm long, 0.58 mm high). Postero-dorsal margin of female carapace straight, with distinctive collar-like fold in right valve. Anterior margin broadly rounded, posterior margin with angular apex. Ventral margin slightly concave two-fifths of length from anterior end. Postero-dorsal flap of LV longer than that of male (Fig. 5).

A2 (Fig. 21) four-segmented. First (Pr) and second (EI) podomeres similar to those of male. Third podomere (EII + EIII) with claws G1, G2, and z1; short seta G3; longer setae z2, and z3; mid-dorsal seta; one postero-ventral aesthetasc y2; and four mid-ventral setae (t1-4). Fourth podomere (EIV) with GM, shorter GM, one apical seta, and aesthetasc y3.

Palp of L5 (Fig. 22) simple, non-segmented, with three apical setae.

FRO (Fig. 23) with elongate, somewhat rounded posterior projection with digitiform end. Spiral canal situated anteriorly, with wall thickened near seminal receptacle. Vaginal opening inconspicuous, without rimmed chitinized ring.

In other characters, female similar to male.

Diagnosis

Carapace with dorsal hump one-third of length from posterior end. Male carapace has anterior and posterior margins equally rounded. Female carapace has straight postero-dorsal margin, with distinctive collar-like fold in right valve. Male hemipenis very large; medial lobe elongate, rounded distally; outer lobe elongate, with spine-like protrusion; both lobes toward dorsal side. M-process well developed, S-shaped, with rounded distal end. Projection on female reproductive organ elongate, tapering distally.

Etymology

The specific epithet is an adjective derived from Kushiro Marsh, type locality, in combination with the Latin suffix -ensis.

Taxon discussion

Fabaeformiscandona kushiroensis sp. nov. clearly belongs in the acuminata group ( Meisch 2000), having four setae in the setal group on the second segment of the Md palp. In east Asia, the acuminata group contains four species, F. danielopoli Yin and Martens 1997, F. nishinoae Smith and Janz 2008, F. akaina Smith and Janz 2008 and Candona quasiakaina Karanovic and Lee 2012. Although the new species from Kushiro Marsh is generally similar to these four Asian species, these five species are clearly distinguished form each other by the following characters. 1) The posterior dorsal margin of the female RV [almost straight with a conspicuous inner fold in F. kushiroensis ; broadly rounded in the other four species]; 2) the medial lobe (h) of Hp [medial lobe elongate and directed in the dorsal direction in F. kushiroensis ; relatively small and distally digitiform in F. danielopoli ; distally flat in F. nishinoae ; distally square-shaped in F. akaina ; distally large and square-shaped in C. quasiakaina ;]; 3) M-process of Hp [distally rounded in F. kushiroensis ; distally digitiform in the other four species]; 4) FRO [with elongate, distally digitiform process in F. kushiroensis ; with elongate, proximally somewhat broad process in F. danielopoli , F. nishinoae and F. akaina ; with relatively short, proximally stout process in C. quasiakaina ]. It is generally similar to F. hyalina ( Brady and Robertson 1870) and F. levanderi ( Hirschmann 1912) in the shapes of the female and male valves, respectively. However, these three species differ in 1) the postero-dorsal margin of the female RV [almost straight with a conspicuous inner fold in F. kushiroensis ; almost straight in F. hyalina ; broadly rounded in F. levanderi ]; 2) the distal lobes of Hp [outer (a) and medial (h) lobes elongate and directed in the dorsal direction in F. kushiroensis ; outer lobe similar to F. kushiroensis , medial lobe stout and broad in F. hyalina ; outer lobe broad and partly subdivided, medial lobe lacking in F. levanderi ]; 3) M-process of Hp [distally rounded in F. kushiroensis ; proximally stout and distally digitiform in F. hyalina ; proximally stout and distally inflated in F. levanderi ]; 4) FRO [with elongate, distally digitiform process in F. kushiroensis ; conical in F. hyalina : with elongate, distally rounded process in F. levanderi ]. Karanovic and Lee (2012) pointed out that these four species from east Asia and F. hyalina from Europe were closely related species in which having tapering/pointed tip on outer lobe of Hp. The new species form Kushiro Marsh also may belong to the group, because of having similar outer lobe of Hp.

Genome structure

Gene contents and organization

The mitochondrial genome of F. kushiroensis is about 17 kbp in size and includes 13 protein-coding genes, 22 tRNA genes, and two rRNA genes, as typically found in other arthropods (Fig. 24, Table 1). The order of the tRNA, rRNA, and protein-cording genes is that of the putative pancrustacean gene order ( Kilpert et al. 2012), with the exception that the position of tRNALys has been translocated to between tRNAAla and tRNAArg (Fig. 25). There is one major non-coding region that presumably contains the origin of replication and regulatory elements for transcription, as well as tandem repeat sequences. There are small gene overlaps at 12 gene borders (Table 1), with the largest (between COIII and tRNAGly) being 50 nucleotides long. Short, non-coding sequences are also present between these genes. The largest spacer sequence (between ND4 and ND4L) is 80 nucleotides long.

Nucleotide composition

The overall A + T content of the F. kushiroensis mitochondrial genome is 68.8% (A = 35.1%; T = 33.7%; G = 11.8%; C = 19.4%), which is relatively low among arthropods. Table 2 gives the A + T content for each type of gene and the CR, and Table 3 gives the nucleotide composition of each codon position for the coding genes.

Long tandem repeat sequences in the CR

The CR contains a long tandem repeat region composed of replicated 220-bp units, three of which were sequenced at each end of the region (Fig. 26). However the sequence of central part of the region is missing due to the technical reasons by Sanger sequencing method. The length of this region is about 2 kbp, estimated from electrophoresis.

Transfer RNA genes

The A + T content of the 22 tRNA genes is 72.2%, which is higher than the overall A + T composition of the mtDNA. The secondary structures of the 22 tRNA genes were determined from the MITOS Web Server to be complete cloverleaves (Suppl. material 2). The anticodon nucleotides for the corresponding tRNA genes are identical to those in V. hilgendorfii . The tRNALys and tRNASer(AGN) genes encode UUU and UCU instead of the more common CUU and GCU, respectively, typical of arthropod mtDNA. Furthermore, the tRNASer(AGN) gene has lost the D-arm, which is also the condition in V. hilgendorfii . Several overlaps associated are with tRNA genes (Table 1).

Ribosomal RNA genes

The 12S rRNA (srRNA) and 16S rRNA (lrRNA) genes in F. kushiroensis are 708 and 1121 nucleotides long, respectively. Their locations and lengths were determined from an analysis of similarity to the V. hilgendorfii homologs and by analysis of their secondary structures reconstructed with Centroidfold (Suppl. material 3).

Protein-coding genes

Initiation codons in the mitochondrial protein-coding genes in F. kushiroensis are ATT (ATP8, COI, COIII, Cyt b, ND3 and ND4L), ATA (ND1, ND2, ND4 and ND5), ATC (COII, ND6), and ATG (ATP6) (Table 2). Twelve of the 13 protein-coding genes terminate with the stop codon TAA, whereas one (ND1) terminates with TAG.

Nuclear ribosomal RNA genes

The nuclear ribosomal RNA genes in F. kushiroensis are determined by a single sequence in which total 7805 nucleotides long, including internal transcribed spacer 1 and 2. Partial 18S rRNA (srRNA), complete 5.8S (lrRNA) and partial 28S rRNA (lrRNA) genes are 1792, 155 and 4007 nucleotides long, respectively. Their locations and lengths were determined from an analysis of similarity to the other sequences of Podocopid ostracods on GenBank and by analysis of their secondary structures reconstructed with Centroidfold.